title: Efficient cultivation conditions for human limbal epithelial cells.
abstract: To compare the stem niche in different culture conditions of limbal epithelial cells, the suspended human limbal epithelial cells (HLECs) were seeded on the 3T3-pretreated plates and the other suspended cells were plated on amniotic membranes (AMs) which were either cryo-preserved or freeze-dried. All were cultured for 10 to 12 days. Reverse transcription-polymerase chain reaction (RT-PCR) for ATP-binding cassette, subfamily G, member 2 (ABCG2), p63, cytokeratin 12, and connexin 43 were performed in cultivated HLECs and their expression levels were compared. The mRNA expression of all markers examined showed no statistically significant differences between the cells on cryo-preserved and on freeze-dried AM. The expression of p63 and cytokeratin 12 in cultivated cells on AMs were significantly lower than those in 3T3-cocultured cells on RT-PCR and immunofluorescent staining. Cultivated HLECs on AMs showed reduced proliferation and differentiation while maintaining stem-property regardless of the preservative method of AM.
authors.affiliation: Department of Ophthalmology, Seoul National University College of Medicine, and Seoul ARtificial Eye Center, Seoul National University Hospital Clinical Research Institute, Seoul, Korea.
authors.forename: Mee Kum
authors.initials: MK
authors.lastname: Kim
authors.forename: Jae Lim
authors.initials: JL
authors.lastname: Lee
authors.forename: Joo Youn
authors.initials: JY
authors.lastname: Oh
authors.forename: Mi Sun
authors.initials: MS
authors.lastname: Shin
authors.forename: Kyeong Seon
authors.initials: KS
authors.lastname: Shin
authors.forename: Won Ryang
authors.initials: WR
authors.lastname: Wee
authors.forename: Jin Hak
authors.initials: JH
authors.lastname: Lee
authors.forename: Ki Sook
authors.initials: KS
authors.lastname: Park
authors.forename: Young Sook
authors.initials: YS
authors.lastname: Son
mesh_terms: D016475:3T3 Cells; D000818:Animals; D018929:Cell Culture Techniques / Q000295:instrumentation* / Q000379:methods*; D002478:Cells, Cultured; D003584:Cytological Techniques; D017931:DNA Primers / Q000737:chemistry; D004847:Epithelial Cells / Q000378:metabolism*; D006801:Humans; D007150:Immunohistochemistry / Q000379:methods; D053551:Keratin-12 / Q000378:metabolism; D051379:Mice; D008954:Models, Biological; D010750:Phosphoproteins / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013234:Stem Cells / Q000166:cytology; D015534:Trans-Activators / Q000378:metabolism
chemical_list: D017931:DNA Primers; D053551:Keratin-12; D010750:Phosphoproteins; D015534:Trans-Activators; C486419:Trp63 protein, mouse